Diseases such as cancer have long been identified by examining tissue biopsies to identify unusual cells. The problem has been that there has been no satisfactory prior-art method to capture a single cell or multiple cells of interest from the surrounding tissue. Currently, investigators must attempt to manually extract, or microdissect, cells of interest either by attempting to mechanically isolate them with a manual tool or through a convoluted process of isolating and culturing the cells. Most investigators consider both approaches to be tedious, time-consuming, and inefficient.
A new technique has been developed which can extract a single cell or a small cluster of cells from a tissue sample in a matter of seconds. The technique is called laser capture microdissection (LCM). In laser capture microdissection, the operator looks through a microscope at a biological specimen such as a tissue biopsy section mounted on a standard glass histopathology slide, which typically contains a variety of cell types. A transfer film is placed over the tissue biopsy section such that the transfer film may or may not contact the tissue. Upon identifying a cell or a group of cells of interest within the tissue section with the aid of a microscope, for example, the operator generates a pulse from a laser. The laser pulse causes localized heating of the thermoplastic film, imparting to it an adhesive property, and thereby, activating the film. The target cells then stick to the localized adhesive area of the thermoplastic film directly above them. Upon removal of the film from the biopsy tissue, the selected cells or sections of tissue are transferred along with the film. Because of the small diameter of the laser beam, extremely small cell clusters or single cells may be microdissected from a tissue section. Biomolecules are then extracted from the transfer film for subsequent analysis.
By taking only these target cells directly from the tissue sample, scientists can immediately analyze the DNA, RNA, proteins, or other biomolecules in order to characterize the activity of the target cells using other research tools. Such procedures as polymerase chain reaction amplification of DNA and RNA, and enzyme recovery from the tissue sample are typically employed.
Laser capture microdissection has successfully extracted cells in many types of tissues. These include kidney glomeruli, in situ breast carcinoma, atypical ductal hyperplasia of the breast, prostatic interepithielial neoplasia, and lymphoid follicles. The direct access to cells provided by laser micro-capture will likely lead to a revolution in the understanding of the molecular basis of cancer and other diseases, helping to lay the groundwork for earlier and more precise disease detection.
Another likely role for the technique is in recording the patterns of gene expression in various cell types, an emerging issue in medical research. For instance, the National Cancer Institute's Cancer Genome Anatomy Project (CGAP) is attempting to define the patterns of gene expression in normal, precancerous, and malignant cells. In projects such as CGAP, laser capture microdissection is a valuable tool for procuring pure cell samples from tissue samples.
The LCM technique is generally described in the published article: Laser Capture Microdissection, Science, Volume 274, Number 5289, Issue 8, pp 998-1001, published in 1996, the entire contents of which are incorporated herein by reference. The purpose of the LCM technique is to provide a simple method for the procurement of selected human cells from a heterogeneous population contained on a typical histopathology biopsy slide.
A typical biological specimen is a tissue biopsy sample consisting of a 5 to 10 micron slice of tissue that is placed on a glass microscope slide using fixation and staining techniques well known in the field of pathology. This tissue slice is a cross section of the body organ that is being studied. The tissue consists of a variety of different types of cells. Often a pathologist desires to remove only a particular cell type or a small portion of the tissue for further analysis. Another typical biological specimen is a layer of cells coated from a liquid suspension.
Laser micro-capture employs a transfer film that is placed over the tissue sample such that it may or may not contact the tissue sample. In contact micro-capture, the transfer film contacts the tissue sample prior to activation by the laser pulse. Due to the friable nature of tissue sections, loose material (whole cell or macromolecular) is likely to adhere to the transfer film even though it was not targeted by the laser. Hence, non-specific transfer of material results. If these non-targeted portions are transferred to the reagent vessel for subsequent analysis, they will be digested by the reagents and contaminate the targeted portions in the sample. Therefore, it is important to prevent the non-targeted portions such as loosely bound tissue areas from contacting the transfer film. Reducing the incidents of non-specific transfer is one aspect of the present invention.
One way of reducing the problem of non-specific transfer is to provide a non-stick barrier layer as described in co-pending application U.S. Ser. No. 09/562,495 filed on May 1, 2000, which is, in its entirety, incorporated herein by reference. Another way of reducing non-specific transfer, for example, is non-contact LCM. In non-contact LCM, the transfer film is offset or distanced a few microns from the tissue sample as described in co-pending application U.S. Ser. No. 08/984,979 filed on Dec. 4, 1997, which is, in its entirety, incorporated herein by reference. As described in this co-pending application, stand-offs are employed to distance or offset the transfer film a few microns from the tissue sample. Distancing the transfer film from the tissue sample reduces incidents of non-specific transfer. However, if stand-offs are employed, non-specific transfer of material is generally confined to the stand-off portions that generally contact the tissue sample in order to space the transfer film away from the tissue sample. Since the stand-off portions are loci for non-specific transfer, it is desirable to prevent the contamination of targeted cells with non-specific material from the stand-off portions. Contamination is particularly possible when extraction fluids such as buffer is introduced to contact the desired material on the transfer film for extraction of particular biomolecules and their subsequent analysis. If buffer is brought into contact with non-specific material on the transfer film such as the material on stand-off portions, that material will be digested along with the targeted material and thereby contaminate the analysis. Therefore, it is desirable to prevent the incorporation of non-specific material. The present invention is aimed at reducing non-specific transfer of material that would contaminate the analysis. Also, the present invention facilitates the introduction of extraction fluids for the post-LCM extraction of desired biomolecules.